Kinetics of iron passage through subcellular compartments of rabbit reticulocytes

Summary The kinetics of the separate processes of Fe₂(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and⁵⁹Fe,¹²⁵I-labeled rabbit transferrin. The binding of⁵⁹Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×10⁵ m ^–1 sec^–1. The rate of internalization of⁵⁹Fe-transferrin was directly measured at 520±100 molecules of Fe₂(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe₂(III)-transferrin internalization the flux of⁵⁹Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec^–1 and 0.0343±0.0118 sec^–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec^–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe₂(III)-transferrin that are distinct from the handling of transferrin.     

Diketopinic acid — A novel reagent for the modification of arginine

Diketopinic acid has been synthesized and shown to be a reagent of choice for specific, reversible modification of the guanidine groups of arginine residues. Diketopinic acid is a yellow crystalline substance and the carboxyl group of the reagent is a convenient handle for attachment to other molecules. The adducts of diketopinoyl derivatives with the guanidine group are cleaved by 0.2 M o-phenylenediamine at pH 8–9. The modification and regeneration of arginine and of arginyl residues in soyabean trypsin inhibitor and insulin are presented as demonstrations of the use of the reagent. The use of diketopinoyl resin in the separation of oxidized A and B chains of insulin has been discussed. Presented in part at the Ninth American Peptide symposium 1985, Toronto, Canada.     

Potassium Fluxes in Chlamydomonas reinhardtii (II. Compartmental Analysis)

42K+ and 86Rb+ were used to determine the subcellular distribution of potassium in Chlamydomonas reinhardtii by compartmental analysis. In both wild type and a mutant strain, three distinct compartments (referred to as I, II, and III) were apparent. Using 42K+, we found that these had half-lives for K+ exchange of 1.07 min, 12.8 min, and 2.9 h, respectively, in wild-type cells and 0.93 min, 14.7 min, and 9.8 h, respectively, for the mutants. Half-lives were not significantly different when 86Rb+ was used to trace K+. Compartments I and II probably correspond to the cell wall and cytoplasm, respectively. Based on the lack of a large central vacuole in Chlamydomonas, the effect of a dark pretreatment on the kinetic properties of compartment III and the similarity between the [K+] of compartment III and that of isolated chloroplasts, this slowly exchanging compartment was identified as the chloroplast. Growth of wild-type cells at 100 [mu]M (instead of 10 mM K+) caused no change of cytoplasmic [K+] but reduced chloroplast [K+] very substantially. The mutants failed to grow at 100 [mu]M K+.     

Kinetics of NO3- Influx in Spruce

Influxes of 13NO3- across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Three kinetically distinct uptake systems for NO3- were identified. In seedlings not previously exposed to external NO3-, a single Michaelis-Menten-type constitutive high-affinity transport system (CHATS) operated in a 2.5 to 500 [mu]M range of external NO3- [NO3-]o. The Vmax of this system was 0.1 [mu]mol g-1 h-1, and the Km was approximately 15 [mu]M. Following exposure to NO3- for 3 d, this CHATS activity was increased approximately 3-fold, with no change of Km. In addition, a NO3--inducible high-affinity system became apparent with a Km of approximately 100[mu]M. The combined Vmax for these discrete saturable components was 0.7 [mu]mol g-1 h-1. In both uninduced and induced plants a linear low-affinity system, additive to CHATS and an NO3--inducible high-affinity system, operated at [NO3-]o [greater than or equal to] 1 mM. The time taken to achieve maximal rates of uptake (full induction) was 2 d from 1.5 mM [NO3-]o and 3 d from 200 [mu]M [NO3-]o.     

Transfection,expression, and DNA sequence of a gene encoding a BoLA-A11 antigen

The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence databases, and have been assigned the accession numbers X82671–X82675     

In vivo cloning of a carboxy-terminalrpoB allele which confers altered transcriptional properties

A 3′-terminal mutation of the gene encoding the β subunit ofEscherichia coli RNA polymerase was isolated using anin vivo polA(Ts) technique. Cloning of the allele was monitored by virtue of the fact that the deletion Δ(rpoB) 1570-1 resulted in an altered-size restriction fragment. DNA sequencing confirmed the predicted nature and location of the mutation: Δ(rpoB) 1570-1 involved an in-frame deletion of 186 bp (62 codons) encoding amino acid residues 967–1028. The phenotype conferred by Δ(rpoB) 1570-1 is discussed with respect to conserved domains within the β polypeptide. Dedicated to Dr. J. Spížek on the occasion of his 60th birthday     

The effects of temperature and hyperoxia on arterial PO2 and acid-base status in Piaractus mesopotamicus

The effects of hyperoxia and change of temperature (range 20–30° C) on blood gases were studied in the teleost fish Piaractus mesopotamicus , native to several major river systems in Brazil. Large hyperoxia-induced increases of arterial P o₂ ( P _ao₂) indicated that true branchial blood shunts are negligible in relation to total gill perfusion. This implies that blood gases will be influenced by ventilation rather than by shunts. Acute variations of temperature ( t ) were accompanied by changes of arterial blood pH (on the average Δ p H_aΔt⁻¹ of — 0·015 units °C⁻¹), due mainly to alterations of P _aco₂: 2·4 mmHg at 20° C, 5·0 mmHg at 30° C. Concomitantly, P _ao₂ declined from 116 mmHg (20° C) to 89 mmHg (30° C). The data suggest that temperature-induced changes of acid-base status depend mainly on gill ventilation and that the decrease of P _ao₂ with higher temperature is a result of this regulation.     

Reconstitution of the transferrin receptor in lipid vesicles. Effect of cholesterol on the binding of transferrin

Purified rabbit reticulocyte transferrin receptors were incorporated into phosphatidylcholine vesicles containing varying amounts of cholesterol. The binding of transferrin to the receptor in the reconstituted vesicles had three distinct characteristics: (1) The binding of transferrin exhibited the two components characteristic of transferrin binding to erythroid cells, a saturable, specific component and a nonsaturable, nonspecific component. (2) Transferrin binding exhibited positive cooperativity at low cholesterol/phospholipid (C/P) molar ratios. However, the cooperativity diminished and then disappeared as the C/P molar ratios were increased to the levels found in circulating red blood cells. (3) The amount of specific transferrin binding to the reconstituted vesicles also decreased as the C/P molar ratio was increased. These results indicate that in the reconstituted system the lipid environment plays a significant role in the expression of transferrin receptors.